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UNCLASSIFIEDMARTIN MARIETTA ENERGY SYSTEMS LIBRARIES

ORNL 688alth & Biology

Oa

3 M4Sb D3t,0S0fl 1

STUDIES ON THE MECHANISM OF THE OXYGEN

EFFECT ON THE RADIOSENSITIVITY

OF TRADESCANTIA CHROMOSOMES

OAK RIDGE NATIONAL LABORATORY

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Report Number: 0RNL- 688Health and BiologyThis document consists of 17pages.

Copy 7 of loQ Series A

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Contract No. W-7405-Eng-26

BIOLOGY DIVISION

STUDIES ON THE MECHANISM OF THE OXYGEN EFFECT ON THE

RADIOSENSITIVITY OF TRADESCANTIA CHROMOSOMES

Norman H. Giles, Jr. and Herbert Parkes Riley

DATE ISSUED

MAY 2 4 1950

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2a

STUDIES CH THE MECHANISM OF THE OXYGEN EFFECT ON THE

RADIOSENSITIVITY OF TRAPESCANTIA CHROMOSOMES*

U Ssnafi 1* {aieB- &• Sflia 3SE&SE& ££&£& Bilss**Biology Division, Oak Ridge Rational Laboratory

Previous experiments (Giles and Riley1) have demonstrated that the radiosen-

sitivity of Ttradeseantia chromosomes, as measured by the occurrence of B»ray

induced aberrations in microspores, is markedly influenced by the amount of oxygen

present. The frequencies of both interchanges and interstitial deletions observedfour to five days following treatment are reduced if inflorescences are irradiated

in gases such as nitrogen and helium and increased if exposures are made in pure

oxygen instead of in air. On the basis of these experiments it was not possible

to decide whether this effect of oxygen is exerted by way of the initial breakage

mechanism, such that more breaks are produced by Xrays In the presence of oxygen,

or whether the effect is on the recovery process, such that new reunions of broken

ends are favored over restitutions, She present paper will discuss experiments

performed to investigate this problem. This evidence indicates that the effeot

of oxygen is on the initial breakage mechanism rather than on the recovery process.

Additional data will also be presented on the relation between aberration frequency

and the percentage of oxygen present at the time of irradiation.

Experimental Methods.. — Inflorescences of Tradescantia ssiiMSSA Anders, and

Woodson, clone 5(unless otherwise noted) of Sax were used. Acetocarmine smear

preparations of microspores at the first postmeiotic mitosis were made on thefourth and fifth daysfollowing irradiation and slides were scored for chromosomal

aberrations — interchanges (dicentrics and centric rings) and interstitial

deletions. In general, 50 or 100 cells from three to eight slides (each from a

separate inflorescence) were scored and standard errors were calculated as

previously* The same X-ray source was utilized — a Coolidge self-rectifying

tube with tungsten target, operated at 250 kv and 15 ma. The inherent filtration

was equivalent to 3 mm. of aluminum.

Irradiations were carried out as in earlier experiments in a lucite exposure

chamber. However, in order to insure a more adequate control of the gas in the

chamber and to facilitate a rapid removal or introduction of gas the experimental

apparatus was redesigned. The new exposure chamber was placed directly in the

X-ray machine and attached by pressure tubing and appropriate stopcock arrange

ments to a vacuum pumpp a gas cylinder, and a mercury manometer. With this

apparatus, all evacuations and introductions of gases could be performed directly

with the inflorescences inside the exposure chamber. Evidence will be presented

that the pre-exposure evacuations in a suction flask, as carried out in the earlier

experiments;, are unnecessary0 Further, it is possible with this equipment to

irradiate in a vacuum or under pressure and to introduce or remove gases during

the period of irradiation. The presence of a manometer makes it possible to

reproduce evacuation conditions and to detect possible unexpected changes of pres

sure in the system.

Results and Discussion,, — A preliminary series of experiments was performed

to determine whether the pre-exposure evacuations in a suction flask, as carried

out in earlier experiments;, were necessary to remove air enclosed around the

anthers by the sepals and petals of the buds. For these tests two sets of buds

were evacuated five times in a flask and helium permitted to diffuse in before

irradiation. One set was placed in the exposure chamber (the original chamber

was used), which was then evacuated and helium admittedjthe other set was placed

in the exposure chamber in air. The third set of buds was not pre-evacuated, but

placed directly in the exposure chamber, which was then evacuated and helium

admitted. All three sets received 400 r at 50 r/min. (table 1). It is clear that

Comparative Effects pf. Various Pretreatments and, Exposure-Chamber

Conditions on. ihg fiyyuenjgjr o£ X-Rav Induced ChromogomA^

Rearran^ejjen^g jg lESigSSfifiJ&B MigfiglfiSESfi

400 r at 50 r/min.later-

Exposure No,. Inter- Satarchanges atitial kfift £fl£•PtWirffi* Conditions C£ls. chjggej Egg g^ fiejj^onj Cg£

Buds pre-evacu- Helium in 226 55 0.24 * 0.03 51 0.23 * 0.03ated and helium chamberadmitted

Bud* TTP-evacu- Air in 400 289 0.72 * 0.04 323 0.81 * 0.05ated and helium chamberadmitted

None Helium in 300 73 0.24 * 0.03 72 0.24 * 0.03chamber

the pretreatment has no effect on the frequency of aberrations, but rather that

the gas in which the exposure is made is the important faetor. Evidently evacuation

in the exposure chamber is sufficient to effect gas exchange in the buds. On the

basis of these results, pre-evaeuation of buds with a water pump was discontinued.

Further;, with the new chamber and a vacuum pump it was possible to evacuate

directly to considerably lower pressures than in the earlier experiments.

In the original experiments comparative exposures were made at only three

oxygen levels — in nitrogen or helium (no oxygen), in air (ea-21$ oxygen), and in

pure oxygen. It seemed of considerable interest to obtain further data in order

to determine the quantitative relation between aberration frequency and percentage

of oxygen during irradiation. Consequently, two separate experiments were carried

out (in one clone 5 was used, in the other, clone 3 of Sax) in which exposures to

a single X-ray dose — 400 r at 50 r/min. — were made with seven different per~

centages of oxygen in the lucite chamber. The oxygen percentages were as follows%

0$ (irradiation in pure — 99.8$ — helium); 2$ (* 98$ helium)$ 10$ (* 90$ helium);

21$ (air); 60$ (*- 40$ helium); 99.5$ (pure oxygen from a commercial cylinder); and

pure oxygen at an absolute pressure of 1500 am. of mercury (approximately 760 an*

above normal atmospheric pressure at Oak Ridge). Before each exposure, inflores

cences were plaoed la the chamber, which was then evacuated to approximately 1 to

2 ma. of mercury, and the particular gas or gas mixture admitted. This procedure

was repeated five times. Following irradiation, the lafloreseenoes remained In

the chamber for approximately ten minutes in the same gas and were then removed

to air. The results of these experiments are presented in table 2 and figure 1.

There is good agreement between the two experiments except for the two points in

air, where, for some unexplained reason, the values obtained for clone 3 are

considerably higher than expected. The data indicate that there is a very rapid

rise in aberration frequency between 2 and 20$ oxygen, after which a gradual

e 2s^

Egfgct of Various Percentage^ gf Oxygen og th£ Frequency £f CJgoj^sjjjflsJ,

rrations la Tradescantj

4^ X^jlay, Exposures of 400 r at 50 r/ndn,.

en Clone M° M» Interchanges. Interstitial

LeticPercentage Used

Clone 3

Cells

450

Interchanges Per. Cell

0 120 0.27 i 0.02

Clone 5 400 113 0.28 ± 0.03

2 Clone 3 284 73 0.26 ± 0.03

Clone 5 315 82 0.26 * 0.03

10 Clone 3 425 304 0.72 * 0.04

Clone 5 200 152 0.76 ± 0.06

21 ©lone 3 350 322 0.92 ± 0.05

(air) Clone 5 150 118 0.79 * 0.07

60 Clone 3 303 283 0.93 * 0.06

Clone 5 200 181 0.91 * 0.07

100 Clone 3 250 249 1.00 ± 0.06

Clone 5 150 148 0.99 ± 0.08

100 Clone 3 225 245 1.09 * 0.07(at an absolute pressureof 1500 mm. Hg )

?er Ce

104 0.23 *• 0.02

88 0.22 ±0.02

95 0.33 * ©.03

77 0.24 * 0.03

305 0.72 * 0.04

162 0.81 x 0.06

392 1.12 * 0.06

116 0.77 * 0.07

364 1.20 * 0.06

196 0.98 * 0.07

318 1.27 * 0.07

159 1.06 i 0.08

279 1.24 * 0.07

Figure 1. Relation between percentage of oxygen in exposure chamber

and frequency of chromosomal interchanges per cell in

Tradesoantia microspores. All exposure to one X-ray dosage •

400 r at 50 r/min. Two separate experiments, one with clone

3 and one with clone 5, as indicated.

_JLJO

or

uj 1.2-CL

(f>UJo

<XoorUJ

SQ8

.0-

oSQ 0.6-

oor

5 0.4-1

Q2-

0-

400r AT50r/MIN.

A EXP. I- CLONE 3

0 EXP 2- CLONE 5

"A A~ -Tr-

lOA—

20(AIR)

30 40 50—7T~

60 70

PER CENT OXYGEN IN EXPOSURE CHAMBER

80 90" A "

1001 \r

o

>03WM

M

O

a

s;

co-a

100% 02 ATABSOLUTE PRESSUREOF 1500mm Hg-

10

increase apparently occurs. The significance of the fact that essentially the

same aberration frequencies were obtained in exposures made in pure (99.8$) helium

and 2% oxygen (f 3&% helium) is not yet clear. This may mean that not all of the

dissolved air is removed from the tissues by the evacuation procedure. It is also

desirable to check further the accuracy of the reported percentage of oxygen in the

gas mixture used (obtained from a commercial source). The general problem of the

effect of oxygen during irradiation at low oxygen tensions is being investigated

further.

The major problem requiring further investigation was concerned with the

mechanism of the oxygen effect in increasing aberration frequencies — whether

this effect resulted from a higher Initial production of chromosome breaks, or

from a relative increase in new reunions as opposed to restitutions of broken ends

during the recovery process. It is clear from the earlier studies of Sax.,2 Marinelli,

Nebelj, Giles and Charles,^ and Lea and Catcheslde/" that in Tradesoantia there is

an appreciable time interval between the production of a break and its disappearance,

either by restitution or new reunion (Interchange). The average time of restitution

for the majority of the breaks has been estimated by Lea^ to be about four minutes.

Thus it would appear to be experimentally feasible to determine whether the oxygen

effect is on initial breakage or on recovery if these two processes can be made to

take place under different conditions with respect to the presence or absence of

oxygen. The following series of comparative exposures (all at a single constant

dosage of 300 r at 300 r/min.) was accordingly carried out. The exposure conditions

about to be described are summarized in table 3. In series 1 and 2 buds were

evacuated for five minutes at 1 to 2 mm. of mercury and irradiated an vacuum.

Series 1 was maintained in vacuum in the exposure chamber for ten minutes following

irradiation. In series 2 pure oxygen was introduced into the exposure chamber to

an absolute pressure of 1500 mm, of mercury immediately following irradiation. The

1

2

[

t

ts nTn»a^ntlt»g that the Effect of Oxygen in Increasing the R^iflg»n«ltivity^ Trafleacantia Chromosomes is on the ^git^ &££&& jegjanig*, Rather,

than ofi the, Reunion Process. && Exposures', 300 r at 300 r/min.

Bncls in Vacuum

Buds in Vacuum

Buds in Oxygen

Buds in Oxygen

Beds in Vacuum

Buds in Oxygen

E

Conditions

Vacuum

Vacuum

Oxygenat 1500 mm.

of Hg

Oxygen at1500 mm. of

Hg

1st 30 seesvacuum. 2nd30 sees oxygenintroduced

(within 3 sec.)to 1500 mm. of Hg

1st 30 sees Oxygen introducedoxygen at 1500 (within 3 sec) toam. of Hg. 2nd 1500 mm. of Hg -30 sees evacuated 10 min.(within 25 sec.)te 1 to 2 mm. of

Hg

Posttreatment

londitione Cells

880

700

Jn£e£j|hjnj|ej|Per Ce]

0.12 * 0.01

0.09 * 0.01

Vacuum - 10min.

Oxygen introduced (within3 sees.) to1500 mm. of Hg-10 min.

Oxygen at 1500mm. of Hg -10 min.

Evacuation (within 25 sec.)| vacuum10 min.

Evacuation (within25 sec.)? vacuum -10 min.

150 0.70 * 0.07

200 0.72 * 0.06

350 0.39 * 0.03

518 0.61 * 0.03

^Interstitial

Beletlons Per Cell

0.11 * 0,01

0.10 * 0.01

0.83 * 0.07

0.85 * 0.07

0.50 ± 0.04

0.59 ± 0.03

H

introduction of oxygen to this pressure was effected within three seconds following

cessation of the irradiation and the buds were maintained in oxygen for ten minutes.

In series 3 and 4 buds were placed in oxygen (by the usual procedure for evacuation

and introduction of gas) at an absolute pressure of 1500 mm. of mercury and irradiated

in oxygen. Series 3 was maintained in oxygen for ten minutes after irradiation.

In series 4 the chamber was evacuated immediately following irradiation. This

evacuation to 1 to 2 mm. of mercury was accomplished within 25 seconds following

the cessation of the irradiation and the vacuum was maintained for ten minutes. In

series 5 buds were evacuated as in series 1 and 2 and irradiation was commenced with

the buds in a vacuum. At the end of 30 seconds of exposure,oxygen was introduced

into the chamber, without interrupting the irradiation, to an absolute pressure of

1500 mm. of mercury (within three seconds) and after the cessation of the total

irradiation time of 60 seconds, the chamber was immediately evacuated to 1 to 2 mm.

of mercury (within 25 seconds) and the buds kept in vacuum for ten minutes. In

series 6 buds were placed in oxygen as in series 3 and 4 and irradiation was commenced

with the buds in oxygen. At the end of 30 seconds of exposure the chamber was

evacuated, without interrupting the irradiation, to 1 to 2 mm. of mercury (within

25 seconds) and after the cessation of the total irradiation time of 60 seconds

oxygen was reintroduced to an absolute pressure of 1500 mm. of mercury (within three

seconds). The exposure of 300 r/min. for one minute was selected to make the total

time of exposure as short as feasible compared to the average time for restitution,

since restitution takes place during the period of irradiation also. The data

obtained from these exposures are presented in table 3.

It is clear from the first comparison (series 1 and 2) that the addition of

oxygen immediately after irradiation does not increase the frequency of aberrations.

Such an increase would be expected if there were an effect of oxygen on the reunion

process. Nor does the removal of oxygen (series 3 and 4) after irradiation result

in a lower aberration frequency. In both comparisons the observed aberration

frequency apparently depends on the presence or absence of oxygen at the time

of irradiation. The additional experiments were included to test this point further

and also to exclude the possibility that the addition or removal of oxygen (to or

from the cells themselves) after irradiation was not accomplished rapidly enough

to detect an effeot on the reunion process if auoh an effeot existed. In series 5,

it is evident that the addition of oxygen during irradiation results in a marked

increase In aberration frequency. The frequency of interchanges -- 0.39 * 0.03

per cell -- is in faot approximately halfway between the values observed following

irradiation in a vacuum (with reoovery in oxygen) 0,09 * 0,01 and irradiation in

oxygen (with reoovery in a vacuum) — 0.72 • 0,07 -- as would be expeeted if inter

change production (above the threshold level of oa. 0,10 per cell) is directly

related to the amount of oxygen present during the period of irradiation. There

is apparently an almost immediate entranoe of a considerable amount of oxygen into

the cells of the anthers at the beginning of the final 30 seoonds of irradiation

(the period of three seoonds indicated is the time required to introduce oxygen

to a pressure of 1500 mm, of merouryj considerably less time is probably required

for the introduction of sufficient oxygen to produce an almost maximal increase in

aberration frequency). In series 6 there is an appreciable decrease in aberration

frequency accompanying the removal of oxygen during the last 30 seoonds of radiation.

The faot that this decrease is not as marked as the increase noted on the addition

of oxygen in series 5 appears to be quite reasonable, since a considerably longer

period is required to evacuate the chamber than to introduce oxygen.

On the basis of these data it is ooncluded that the effeot of oxygen in in

creasing the radioaensitivity of Tradaaaantia chromosomes results from an increased

production of breaks rather than from an effeot on the reoovery prooesi such that

new reunions are favored over restitutions in the presence of this gas. An earlier

preliminary analysis of the differences in slope of dosage curves obtained in air,

in oxygen and in nitrogen (Giles and Rilejr-) suggested that the oxygen effect was

exerted by way of the recovery mechanism. It now appears that these differences

can be more plausibly interpreted on the basis of an intensity effect. Thus, if

there are in fact more breaks produced in the presence of oxygen than in its absence,

when comparative exposures are made where cells receive the same physical dose at

the same intensity in r/min„ either in oxygen or in its absence (e..g,, in helium,

in nitrogen, or in a vacuum), those cells irradiated in oxygen will be effectively

exposed to a higher intensity in terms of breaks produced per minute. It has been

shown by Sax for chromosomal interchanges that as the intensity of X radiation is

decreased the curve relating aberration frequency to dosage approaches linearity.

In the experiments previously reported the exponents for the dosage curves decreased

from approximately 1.6 in oxygen to approximately 1,3 in nitrogen.

Summary. -«= Further experiments have been performed on the effect of oxygen in

increasing the radiosensitlvity of Tradesoantia microspore chromosomes. Exposures

of inflorescences to a single constant X=ray doses but in atmospheres containing

seven different percentages of oxygen indicate that there is a rapid rise in aberration

frequency between 2 and 21$6 oxygen, with a gradual increase thereafter. Further

studies are being made to clarify the effect of oxygen at levels between zero and

two per cent. Other experiments have been performed to determine whether the oxygen

effect is exerted by way of the initial breakage mechanism or on the reunion process.

These consisted of comparative exposuresto a single dose of 300 r in one minute of

inflorescences in a vacuum or in oxygen with the addition or removal of oxygen either

immediately after or during part of the irradiation period. The experiments show

that the presence or absence of oxygen during the aetual exposure to X rays rather

than during the postirradlation period is the important factor, thus demonstrating

that the effect of oxygen in increasing the radiosensitlvity of Tradesoantia

Jl,/

chromosomes results from an increased production of initial breaks rather than from

an influence on the reunion of broken ends.

Acknowledgement„ — The authors wish to acknowledge the assistance of Lucille

McMichael Falrchild in certain of these experiments.

* This work was done under Contract Ho. W-7405-Eng-26 for the Atomic Energy

Commission, Oak Ridge, Tennessee,

** Present address: Department of Botany, University of Kentucky, Lexington, Kentucky.

1Giles, N. H., Jr. and Riley, H. P., These Proceedings. ^5, 640 (1949).

2Sax, Karl, Jb^, 2J, 225 (1939).

3 Marinelli, L. D., Hebel, B. R,, Giles, N.H., Jr., and Charles, D. R., Am. J.. Botany.

22, 866 (1942).

4 Lea, D. E. and Catcheside, D. G., £. Genet.. ££,216 (1942).

* Lea, D. E., Actions $£ Radiations on, Living Cj^Lj., Cambridge University Press,

Cambridge, England (1945).

6 Sax, Karl, £oJd Spring Harbor Symposia £uan£. Biol,., 9, 93 (1941) •fink